This invention relates to double-stranded ribonucleic acids (dsRNAs), and their use in mediating RNA interference to inhibit the expression of genes, necessary for replication and pathogenesis of Hepatitis B Virus, in particular in the inhibition of viral polymerase, surface antigen, e-antigen and the X protein. Furthermore, the use of said dsRNAs to treat or prevent chronic liver diseases/disorders, inflammations, fibrotic conditions and proliferative disorders, like cancers, as consequence of Hepatitis B Virus infection, is part of this invention.
The Hepatitis B Virus is a strict hepatotrophic, double-stranded DNA containing virus. Although DNA is the genetic material, the replication cycle involves a reverse transcription step to copy a pregenomic RNA into DNA. In order to accomplish this essential step, the viral-encoded polymerase possesses reverse transcriptase activity. Hepatitis B virus is classified as one member of the Hepadnaviruses and belongs to the family of Hepadnaviridae. The primary infection of adult humans with Hepatitis B Virus causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. About 95% of acute hepatitis resolve without treatment. Those patients, that are not able to overcome the virus infection, suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer. Perinatal transmission from Hepatitis B virus-infected mothers to newborns also leads to chronic hepatitis. The treatment options for chronic Hepatitis B Virus infection are limited and lead only in some cases to complete and lasting remission. Additional clinical and therapeutical complications arise in Hepatitis B Virus patients co-infected with Hepatitis C, Hepatitis D or Human Immunodeficiency Virus.
The Hepatitis B Virus is transmitted via blood or blood products, sperm, vaginal secrets, or saliva. Drug abuse and sexual intercourse are dangerous activities and support spreading the virus. Contact of damaged, mucoid epithelia with contaminated body fluids may be sufficient for infection. Incubation time is between 40 to 200 days. The risk for infection is proportional to the number of transmitted Hepatitis B Virus particles. Babies are often infected perinatally by their Hepatitis B Virus carrying mother, a major health problem in endemic areas.
About 2 billion people are infected with Hepatitis B Virus and 400 million are chronic carriers. Areas with high prevalence are Africa and South-East Asia, with local accumulation of 20-80% infected persons.
Based on sequence homology, Hepatitis B Viruses are classified into genotypes A-H, with genotypes A-D being the most important ones. Genotype A is frequent in North-Western Europe, USA, South and Central America. Genotype B and C are dominant in China, Japan, Indonesia and other countries in East Asia. Genotype D is found in Southern Europe, Northern Africa and South Africa. Disease progression and response to pharmaceutical treatment differ among genotypes.
Infectious Hepatitis B Virus particles have a diameter of about 42 nm. The outer membrane bilayer contains the large, middle and small surface protein. The cognate hepatocellular receptor for virus surface protein binding and internalization is unknown. Many copies of core protein form a spherical nucleocapsid structure inside the virus particle. Each nucleocapsid carries partial double-stranded DNA as genetic material, together with viral polymerase.
Upon uptake by hepatocytes, the nucleocapsid is transferred to the nucleus and DNA is released. There, the DNA strand synthesis is completed and gaps repaired to give the covalently closed circular (ccc) supercoiled DNA of 3.2 kb. The cccDNA serves as template for transcription of four major viral mRNAs, which are 3.5, 2.4, 2.1 and 0.7 kb long. All mRNAs are 5′-capped and polyadenylated at the 3′-end. There is sequence overlap at the 3′-end between all four mRNAs.
The 3.5 kb mRNA serves as template for core protein and polymerase production. In addition, the same transcript serves as a pre-genomic replication intermediate and allows the viral polymerase to initiate the reverse transcription into DNA. Core protein is needed for nucleocapsid formation. In addition, sequential processing activities transforms some core protein into the secretable e-antigen. The abundance of e-antigen in blood correlates with Hepatitis B Virus replication in liver and serves as important diagnostic marker for monitoring the disease progression.
The 2.4 and 2.1 kb mRNAs carry the open reading frames pre-S1, pre-S2 and S2 for expression of viral large, medium and small surface antigen. The s-antigen is associated with infectious, complete particles. In addition, blood of infected patients also contain non-infectious particles derived from s-antigen alone, free of genomic DNA or polymerase. The function of these particles is not fully understood. The complete and lasting depletion of detectable s-antigen in blood is considered as reliable indicator for Hepatitis B Virus clearance and thus, successful cure.
The 0.7 kb mRNA encodes the X protein. This gene product is important for efficient transcription of viral genes and also acts as a transactivator on host gene expression. The latter activity seems to be important for hepatocyte transformation during development of liver cancer.
Recombinant Hepatitis B Virus s-antigen is used for vaccination. The injection of three doses of formulated s-antigen at day 1, at 4 weeks and at 6 months usually induces a sufficient titer of neutralizing antibodies. Vaccinated patients are protected for 10 years or longer. However, the vaccines are no substitute for therapy.
Patients with acute Hepatitis B Virus infection are not treated due to the high, natural remission rate. However, those patients with detectable s-antigen, e-antigen or viral DNA in the blood for more than 6 months are considered chronically infected. Nucleoside analogs as inhibitors of reverse transcriptase activity are the first treatment option for many patients. Lamivudine, Tenofovir, or Entecavir suppress Hepatitis B Virus replication, sometimes to undetectable levels. Improvement of liver function and reduction of liver inflammation are the most important benefits. However, only few patients achieve complete and lasting remission after the end of treatment. Furthermore, the Hepatitis B Virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients co-infected with Hepatitis B and Human Immunodeficiency Virus. Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
The second treatment option is the administration of interferon-alpha. Here, patients receive high doses of interferon-alpha over a period of 6 months. Depending on the virus genotype, up to 50% of chronic infection are curable. However, the Asian genotype B gives very poor response rates. Co-infection with Hepatitis D or Human Immunodeficiency Virus renders interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
Despite significant advances in the field of Hepatitis B Virus treatment, there remains a need for an agent that can selectively and efficiently silence the gene expression of the virus, blocks replication and subsequently reduces viral burden in chronically infected patients.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The invention provides double-stranded ribonucleic acid molecules (dsRNAs), as well as compositions and methods for inhibiting the expression of the Hepatitis B Virus gene, in particular the expression of the Hepatitis B Virus gene, in a cell, tissue or mammal using such dsRNA. The invention also provides compositions and methods for treating or preventing pathological conditions and diseases caused by the infection of the Hepatitis B Virus such as in chronic liver diseases/disorders, inflammations, fibrotic conditions and proliferative disorders, like cancers.